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Expression of proteoglycan mRNA in patients with painful clicking and chronic closed lock of the temporomandibular joint
Int. J. Oral Maxillofac. Surg. 2005; 34: 656–658 doi:10.1016/j.ijom.2005.02.008, available online at http://www.sciencedirect.com
Research Paper TMJ Disorders
Expression of proteoglycan mRNA in patients with painful clicking and chronic closed lock of the temporomandibular joint
D. I. Paegle1,2, A. Holmlund1,*, A. Hjerpe2 1 Department of Oral and Maxillofacial Surgery, Karolinska Institutet, Box 4064, SE-141 04 Huddinge University Hospital, Huddinge, Sweden; 2Department of Laborative Medicine, Division of Pathology, Karolinska Institutet, Huddinge University Hospital, Huddinge, Sweden
D. I. Paegle, A. Holmlund, A. Hjerpe: Expression of proteoglycan mRNA in patients with painful clicking and chronic closed lock of the temporomandibular joint. Int. J. Oral Maxillofac. Surg. 2005; 34: 656–658. # 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. RT-PCR was used to analyze the expression of a series of mRNAs coding for proteoglycans aggrecan, versican, biglycan, decorin, fibromodulin and also hyaluronan synthase 1 in specimens obtained during discectomy of the temporomandibular joint in patients with unilateral signs and symptoms of chronic closed lock (eight patients) and painful clicking (seven patients). Regarding the disc, aggrecan expression was higher in patients with chronic closed lock. As for the posterior disc attachment specimens, patients with chronic closed lock showed a tendency for higher expression of biglycan and hyaluronan synthase 1. The degradation of matrix in patients with chronic closed lock of the temporomandibular joint seems not to be caused by a reduced synthesis and the degenerative process seen in these patients is one with low turnover similar to the situation in primary osteoarthrosis of hyaline cartilage. The results indicate that any treatment should intervene early in the disease process of chronic closed lock in order to prevent the development of a degenerative process.
Accepted for publication 1 February 2005 Available online 24 March 2005
Temporomandibular joint (TMJ) internal derangement is the most common TMJ affliction treated surgically. From a clinical point of view internal derangement of the TMJ has two expressions: painful clicking (PC) and chronic closed lock (CL) the latter frequently being associated with osteoarthrosis (OA). OA is a chronic, principally noninflammatory degenerative condition of the articular cartilage where one of the earliest changes is a loss of proteoglycans (PGs) from the tissue matrix. In a recent
both tissues, i.e., that none had changes limited only to the disc tissue, indicate that the losses in both tissues are components of the same degenerative process, and it may be that the alterations in the posterior attachment are of pathogenic importance for the development of chronic closed lock. In OA the diminished amount of PGs can depend either on decreased synthesis, increased degradation, or both. During secondary OA, following experimental perforation of the TMJ disc with incon-
0901-5027/060656+03 $30.00/0
study comparing PC and CL patients, the latter showed a substantial loss of proteoglycans from the affected disc8. Through their strongly pryanionic glycosaminoglycan (GAG) side chains, these PGs create a swelling pressure in the tissue, necessary for their ability to distribute point load. We also showed that when the amount of PGs diminished in the TMJ disc, there was a corresponding loss in the posterior disc attachment too. The fact that all patients with CL had such degenerative changes in
# 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Expression of proteoglycan mRNA in patients gruence of the joint surfaces, increased degradation is paramount in the rapidly progressing OA1. On the other hand, in primary OA, the cartilage matrix degradation is usually preceded by enhanced synthesis of PGs, probably as a response to increased load7. Such a response has been demonstrated in an experimental study11 where prior to OA expression of the tibial articular surface, there was an increased production of matrix PGs. However, the disease progression is slow in primary OA and the loss of PGs that occurs concomitantly with the tissue disruption is rather the result of reduced synthesis. In order to elucidate if the lowered concentration of PGs in the TMJ disc and the posterior disc attachment found in our previous study8 is a result of decreased synthesis or increased turnover we studied the expression of a series of mRNAs coding for PGs and PG-related proteins as a mean of monitoring synthetic rates. In this way we studied the expression of the major PGs giving the fibrous cartilage matrix an osmotic pressure— aggrecan and versican—and the collagen fiber associated small PGs—biglycan, decorin and fibromodulin—as well as hyaluronan synthase 1 (HAS-1), hyaluronan being essential for anchoring of the major PGs in the matrix. Material and methods
During TMJ discectomy, disc specimens including the anterior part of the posterior disc attachment were obtained from eight consecutive patients (seven females) with the clinical diagnosis of CL: a case history of sudden painful reduction of mouth opening and the clinical findings of reduced maximum opening (<40 mm) and reduced maximum protrusion (<8 mm) and tomographic signs of OA (erosions, sclerosis or flattening of the articular surfaces). The mean age of patients was 39.8 years (range 20–62). For comparison, biopsies were taken in the same manner as for the CL patients in seven consecutive patients (five females) with the clinical diagnosis of PC: painful TMJ clicking and no radiographic signs of OA on the preoperative tomograms. The mean age of patients was 37.9 years (range 18–66). All patients had been treated nonsurgically for at least 6 months without success. Exclusion criteria for both patient groups were: (i) other TMJ disease, (ii) dentofacial deformity, (iii) major jaw trauma, (iv) previous TMJ surgery, and (v) previous steroid injection in the TMJ. The disc and posterior disc attachment specimens were separated immediately
and stored on RNAlaterTM (Ambion Inc, Austin, Texas, USA) at 20 8C until processed. Preparation of total RNA
In order to evaluate the expression of the genes coding for the PGs under study we extracted the total RNA from cartilage and connective tissue, using RNeasy Kit (QIAGEN Inc, Hilldan, Germany). Thus, the samples were ground in liquid nitrogen, and total RNA was extracted according to the manufacturer’s instructions. The final RNA pellets were resuspended in 30 ml RNase-free water. The total RNA concentrations were measured spectrophotometrically at 260 nm. RNA was stored at 20 8C until further analysis. RT-PCR
One hundred and fifty nanograms of of total RNA from each sample were first reverse-
657
transcribed into cDNA in a total volume of 20 ml, using SuperScript Reverse Transcriptase (Gibco BRL, Carlsbad, Carlifornia, USA) and oligo(dT) primers pdT15–18 (Pharmacia Biotech, Uppsala, Sweden). Optimal cycling parameters were determined in pilot experiments (Table 1). Thus, cycling parameters were varied to avoid saturation of either analyte or reference amplimer. The selected cycle numbers gave relative fluorescence in the 1:3–3:1 range. The PCR amplification of proteoglycans genes sequences was carried out for 35 cycles: 94 8C for 30 s, annealing temperature for 30 s, 72 8C for 1 min (Mastercycler Thermal Cycler, Eppendorf, Germany). cDNA sequences were analyzed by simultaneously amplifying the glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene. This was obtained by adding 37 pmol of each primer for GAPDH to the reaction after the first 3–12 cycles of amplification. The statistical evaluation comprised repeated analysis of variance (ANOVA).
Table 1. Cycling parameters obtained by optimization for different proteoglycans and proteoglycan related genes
aaa Decorin Biglycan Fibromodulin Aggrecan Versican V0 Versican V1 HAS-1
First amplification without GAPDH primer (number of cycles)
Second amplification with GAPDH primer (number of cycles)
6 3 4 12 12 10 12
29 32 31 23 23 25 23
Fig. 1. The expression of proteoglycan mRNA (relative fluorescence with GAPDH as reference) in the disc and posterior disc attachment (PDA) in patients with painful clicking (P) and chronic closed lock (C). 1 = aggrecan; 2 = biglycan; 3 = decorin; 4 = fibromodulin; 5 = HAS-1; 6 = versican.
658
Paegle et al.
A null hypothesis of no difference was rejected at a significance level of p = 0.05. Results
All PG-related genes studied gave distinct amplimer bands, and with the cycle combinations used both analyte and control bands were unsaturated and within the selected ratio interval. The number of cycles necessary to detect analyte cDNAs was generally larger than that needed when analysing cDNAs from cultured cells with the same primers4,6 but of the same magnitude as those used when analyzing similarly prepared cDNA from aortic tissue10. The relative fluorescence values obtained for the studied PG-associated genes are given in Fig. 1. Some of these genes were similarly expressed in the different groups, while in some cases there was considerable inter-individual variability. In the discs there was a significant increase of aggrecan expression (P < 0.03) in the CL group when comparing tissues from PC and CL patients. For biglycan, fibromodulin and versican, there was a considerable inter-individual variability. In the posterior disc attachment tissue the CL group exhibited slightly increased expression of HAS-1 and biglycan although the differences were not significant at the chosen levels. The relative fluorescence values for fibromodulin and versican varied considerably within the groups, a variability that did not seem to correlate to group identity. Discussion
Compared to PC, CL was accompanied by a loss in GAG content and showed macroscopic signs of OA in our previous study8. This is also interesting from the point of view that both patient groups had a wide age span. The CL associated OA is thus a process separate from physiologic aging. The decrease in GAG content seen in both disc and posterior disk attachment in patients with CL could not (with PC as control) be correlated to a decreased mRNA expression in any of the major PGs normally found in these tissues. In fact, the only change in the disc was a relative increase in aggrecan, well in accordance with the hyalinization of this tissue seen in experimental TMJ OA2. The corresponding profile in the posterior disc attachment tissues only differed with regard to a slightly increased biglycan and HAS-1 expression. In CL patients8, synovitis and proliferation of new vessels are common findings and may be associated with the increased expression of
biglycan. The increased expression of HAS-1 may correlate to both increased proportion of vessels and reactive changes in the fibroblasts. The decrease in GAGs seen in CL in our previous study8 may therefore relate to an increased turnover of matrix components rather than a decreased synthesis rate. Early in the development of OA (primary and secondary) both hyaline and fibrous cartilages exhibit an increased rate of synthesis of matrix components. The metabolic activity leading to this appears to vary; in secondary OA both degenerative and reparative processes takes place simultaneously while in primary OA this ‘reactive’ increase in PG synthesis occurs before microscopic lesions are observed11. The mRNA expression profiles in the PC and CL patients in this study were quite similar, and the results do not indicate a significantly increased synthesis as a cause for the decreased PG content shown in our previous study8. The alternative explanation may be that the OA seen in the CL patients is one with low turn-over, similar to the situation in primary OA of hyaline cartilage. In that process there is a longterm weak imbalance between synthesis and break-down, the result being a slow loss of PGs. It has been assumed that PC may precede a CL condition5 but it has also been suggested that OA is a primary cause preceding the internal derangement9. Long-term follow-up of patients with PC has shown that they, in a considerable proportion have developed clinical signs and symptoms of OA3. Thus, it can be speculated that when PC develops into OA this degeneration will primarily affect the disc margins. The rapid dissemination also to the attachments will affect the physical properties of these looser connective tissues. This will weaken the disc attachments which in turn will allow for a displacement of the disc of sufficient magnitude to cause CL. This hypothesis for the development of CL in PC does not preclude other mechanisms in single cases. Thus, one should expect that any degenerative lesion affecting the margins of the disc may also cause CL even when there is no previous history of PC. Our results may also indicate that any treatment should intervene early in the disease process of CL to prevent the development of a degenerative process. Acknowledgements. We wish to acknowledge Dr. Ainars Leonchicks and the A. Kirchenstein Institute (ASLA Ltd.) for performing the RT-PCR analyses.
References 1. Axelsson S, Bjo¨rnsson S, Holmlund A, Kopp S, Hjerpe A. Metabolic turnover of sulphated glycosaminoglycans and proteoglycans in temporomandibular joint cartilages with experimentally induced osteoarthrosis. Acta Odontol Scand 1994: 52: 65–71. 2. Axelsson S, Holmlund A, Hjerpe . An experimental model of osteoarthrosis in the temporomandibular joint of the rabbit. Acta Odontol Scand 1992: 50: 273–280. 3. DeLeeuw R, Boering G, van der Kuul I, Stegenga B. Hard and soft tissue imaging of the temporomandibular joint 30 years after diagnosis of osteoarthritis and internal derangement. J Oral Maxillofac Surg 1996: 54: 1270–1280. 4. Dobra K, Anda¨ng M, Syrokou A, Karamanos NK, Hjerpe A. Differentiation of mesothelioma cells is influenced by the expression of proteoglycans. Exp Cell Res 2000: 258: 12–22. 5. Eriksson L, Westesson P-LL., Rohlin M. Temporomandibular joint sounds in patients with disc displacement. Int J Oral Maxillofac Surg 1985: 14: 428–436. 6. Gulyas M, Dobra K, Hjerpe A. Expression of genes coding for proteoglycans and Wilms’ tumour susceptibility gene 1 (WT1) by variously differentiated benign human mesothelial cells. Differentiation 1999: 65: 89–96. 7. Maroudas AI. Balance between swelling pressure and collagen tension in normal and degenerate cartilage. Nature 1976: 260: 808–809. 8. Paegle DI, Holmlund AS, Hjerpe A. Matrix glycosaminoglycans in the temporomandibular joint disc in patients with painful clicking and chronic closed lock. Int J Oral Maxillofac Surg 2003: 32: 397– 400. 9. Stegenga B, De Bont LGM, Boering G. Osteoarthrosis as the cause of craniomandibular pain and dysfunction: a unifying concept. J Oral Maxillofac Surg 1989: 47: 249–256. 10. Theocharis AD, Tsolakis I, Hjerpe A, Karamanos NK. Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V0. Atherosclerosis 2001: 154: 367–376. 11. Wei L, Svensson O, Hjerpe A. Proteoglycan turnover during development of spontaneous osteoarthrosis in the guinea pigs. Osteoarthritis Cartilage 1998: 6: 410–416. Address: Anders Holmlund Department of Laborative Medicine Division of Pathology Karolinska Institutet Huddinge University Hospital Huddinge Sweden Tel: +46 85 858 3953 Fax: +46 8 774 3875 E-mail: [email protected]
Research Paper TMJ Disorders
Expression of proteoglycan mRNA in patients with painful clicking and chronic closed lock of the temporomandibular joint
D. I. Paegle1,2, A. Holmlund1,*, A. Hjerpe2 1 Department of Oral and Maxillofacial Surgery, Karolinska Institutet, Box 4064, SE-141 04 Huddinge University Hospital, Huddinge, Sweden; 2Department of Laborative Medicine, Division of Pathology, Karolinska Institutet, Huddinge University Hospital, Huddinge, Sweden
D. I. Paegle, A. Holmlund, A. Hjerpe: Expression of proteoglycan mRNA in patients with painful clicking and chronic closed lock of the temporomandibular joint. Int. J. Oral Maxillofac. Surg. 2005; 34: 656–658. # 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. RT-PCR was used to analyze the expression of a series of mRNAs coding for proteoglycans aggrecan, versican, biglycan, decorin, fibromodulin and also hyaluronan synthase 1 in specimens obtained during discectomy of the temporomandibular joint in patients with unilateral signs and symptoms of chronic closed lock (eight patients) and painful clicking (seven patients). Regarding the disc, aggrecan expression was higher in patients with chronic closed lock. As for the posterior disc attachment specimens, patients with chronic closed lock showed a tendency for higher expression of biglycan and hyaluronan synthase 1. The degradation of matrix in patients with chronic closed lock of the temporomandibular joint seems not to be caused by a reduced synthesis and the degenerative process seen in these patients is one with low turnover similar to the situation in primary osteoarthrosis of hyaline cartilage. The results indicate that any treatment should intervene early in the disease process of chronic closed lock in order to prevent the development of a degenerative process.
Accepted for publication 1 February 2005 Available online 24 March 2005
Temporomandibular joint (TMJ) internal derangement is the most common TMJ affliction treated surgically. From a clinical point of view internal derangement of the TMJ has two expressions: painful clicking (PC) and chronic closed lock (CL) the latter frequently being associated with osteoarthrosis (OA). OA is a chronic, principally noninflammatory degenerative condition of the articular cartilage where one of the earliest changes is a loss of proteoglycans (PGs) from the tissue matrix. In a recent
both tissues, i.e., that none had changes limited only to the disc tissue, indicate that the losses in both tissues are components of the same degenerative process, and it may be that the alterations in the posterior attachment are of pathogenic importance for the development of chronic closed lock. In OA the diminished amount of PGs can depend either on decreased synthesis, increased degradation, or both. During secondary OA, following experimental perforation of the TMJ disc with incon-
0901-5027/060656+03 $30.00/0
study comparing PC and CL patients, the latter showed a substantial loss of proteoglycans from the affected disc8. Through their strongly pryanionic glycosaminoglycan (GAG) side chains, these PGs create a swelling pressure in the tissue, necessary for their ability to distribute point load. We also showed that when the amount of PGs diminished in the TMJ disc, there was a corresponding loss in the posterior disc attachment too. The fact that all patients with CL had such degenerative changes in
# 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Expression of proteoglycan mRNA in patients gruence of the joint surfaces, increased degradation is paramount in the rapidly progressing OA1. On the other hand, in primary OA, the cartilage matrix degradation is usually preceded by enhanced synthesis of PGs, probably as a response to increased load7. Such a response has been demonstrated in an experimental study11 where prior to OA expression of the tibial articular surface, there was an increased production of matrix PGs. However, the disease progression is slow in primary OA and the loss of PGs that occurs concomitantly with the tissue disruption is rather the result of reduced synthesis. In order to elucidate if the lowered concentration of PGs in the TMJ disc and the posterior disc attachment found in our previous study8 is a result of decreased synthesis or increased turnover we studied the expression of a series of mRNAs coding for PGs and PG-related proteins as a mean of monitoring synthetic rates. In this way we studied the expression of the major PGs giving the fibrous cartilage matrix an osmotic pressure— aggrecan and versican—and the collagen fiber associated small PGs—biglycan, decorin and fibromodulin—as well as hyaluronan synthase 1 (HAS-1), hyaluronan being essential for anchoring of the major PGs in the matrix. Material and methods
During TMJ discectomy, disc specimens including the anterior part of the posterior disc attachment were obtained from eight consecutive patients (seven females) with the clinical diagnosis of CL: a case history of sudden painful reduction of mouth opening and the clinical findings of reduced maximum opening (<40 mm) and reduced maximum protrusion (<8 mm) and tomographic signs of OA (erosions, sclerosis or flattening of the articular surfaces). The mean age of patients was 39.8 years (range 20–62). For comparison, biopsies were taken in the same manner as for the CL patients in seven consecutive patients (five females) with the clinical diagnosis of PC: painful TMJ clicking and no radiographic signs of OA on the preoperative tomograms. The mean age of patients was 37.9 years (range 18–66). All patients had been treated nonsurgically for at least 6 months without success. Exclusion criteria for both patient groups were: (i) other TMJ disease, (ii) dentofacial deformity, (iii) major jaw trauma, (iv) previous TMJ surgery, and (v) previous steroid injection in the TMJ. The disc and posterior disc attachment specimens were separated immediately
and stored on RNAlaterTM (Ambion Inc, Austin, Texas, USA) at 20 8C until processed. Preparation of total RNA
In order to evaluate the expression of the genes coding for the PGs under study we extracted the total RNA from cartilage and connective tissue, using RNeasy Kit (QIAGEN Inc, Hilldan, Germany). Thus, the samples were ground in liquid nitrogen, and total RNA was extracted according to the manufacturer’s instructions. The final RNA pellets were resuspended in 30 ml RNase-free water. The total RNA concentrations were measured spectrophotometrically at 260 nm. RNA was stored at 20 8C until further analysis. RT-PCR
One hundred and fifty nanograms of of total RNA from each sample were first reverse-
657
transcribed into cDNA in a total volume of 20 ml, using SuperScript Reverse Transcriptase (Gibco BRL, Carlsbad, Carlifornia, USA) and oligo(dT) primers pdT15–18 (Pharmacia Biotech, Uppsala, Sweden). Optimal cycling parameters were determined in pilot experiments (Table 1). Thus, cycling parameters were varied to avoid saturation of either analyte or reference amplimer. The selected cycle numbers gave relative fluorescence in the 1:3–3:1 range. The PCR amplification of proteoglycans genes sequences was carried out for 35 cycles: 94 8C for 30 s, annealing temperature for 30 s, 72 8C for 1 min (Mastercycler Thermal Cycler, Eppendorf, Germany). cDNA sequences were analyzed by simultaneously amplifying the glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene. This was obtained by adding 37 pmol of each primer for GAPDH to the reaction after the first 3–12 cycles of amplification. The statistical evaluation comprised repeated analysis of variance (ANOVA).
Table 1. Cycling parameters obtained by optimization for different proteoglycans and proteoglycan related genes
aaa Decorin Biglycan Fibromodulin Aggrecan Versican V0 Versican V1 HAS-1
First amplification without GAPDH primer (number of cycles)
Second amplification with GAPDH primer (number of cycles)
6 3 4 12 12 10 12
29 32 31 23 23 25 23
Fig. 1. The expression of proteoglycan mRNA (relative fluorescence with GAPDH as reference) in the disc and posterior disc attachment (PDA) in patients with painful clicking (P) and chronic closed lock (C). 1 = aggrecan; 2 = biglycan; 3 = decorin; 4 = fibromodulin; 5 = HAS-1; 6 = versican.
658
Paegle et al.
A null hypothesis of no difference was rejected at a significance level of p = 0.05. Results
All PG-related genes studied gave distinct amplimer bands, and with the cycle combinations used both analyte and control bands were unsaturated and within the selected ratio interval. The number of cycles necessary to detect analyte cDNAs was generally larger than that needed when analysing cDNAs from cultured cells with the same primers4,6 but of the same magnitude as those used when analyzing similarly prepared cDNA from aortic tissue10. The relative fluorescence values obtained for the studied PG-associated genes are given in Fig. 1. Some of these genes were similarly expressed in the different groups, while in some cases there was considerable inter-individual variability. In the discs there was a significant increase of aggrecan expression (P < 0.03) in the CL group when comparing tissues from PC and CL patients. For biglycan, fibromodulin and versican, there was a considerable inter-individual variability. In the posterior disc attachment tissue the CL group exhibited slightly increased expression of HAS-1 and biglycan although the differences were not significant at the chosen levels. The relative fluorescence values for fibromodulin and versican varied considerably within the groups, a variability that did not seem to correlate to group identity. Discussion
Compared to PC, CL was accompanied by a loss in GAG content and showed macroscopic signs of OA in our previous study8. This is also interesting from the point of view that both patient groups had a wide age span. The CL associated OA is thus a process separate from physiologic aging. The decrease in GAG content seen in both disc and posterior disk attachment in patients with CL could not (with PC as control) be correlated to a decreased mRNA expression in any of the major PGs normally found in these tissues. In fact, the only change in the disc was a relative increase in aggrecan, well in accordance with the hyalinization of this tissue seen in experimental TMJ OA2. The corresponding profile in the posterior disc attachment tissues only differed with regard to a slightly increased biglycan and HAS-1 expression. In CL patients8, synovitis and proliferation of new vessels are common findings and may be associated with the increased expression of
biglycan. The increased expression of HAS-1 may correlate to both increased proportion of vessels and reactive changes in the fibroblasts. The decrease in GAGs seen in CL in our previous study8 may therefore relate to an increased turnover of matrix components rather than a decreased synthesis rate. Early in the development of OA (primary and secondary) both hyaline and fibrous cartilages exhibit an increased rate of synthesis of matrix components. The metabolic activity leading to this appears to vary; in secondary OA both degenerative and reparative processes takes place simultaneously while in primary OA this ‘reactive’ increase in PG synthesis occurs before microscopic lesions are observed11. The mRNA expression profiles in the PC and CL patients in this study were quite similar, and the results do not indicate a significantly increased synthesis as a cause for the decreased PG content shown in our previous study8. The alternative explanation may be that the OA seen in the CL patients is one with low turn-over, similar to the situation in primary OA of hyaline cartilage. In that process there is a longterm weak imbalance between synthesis and break-down, the result being a slow loss of PGs. It has been assumed that PC may precede a CL condition5 but it has also been suggested that OA is a primary cause preceding the internal derangement9. Long-term follow-up of patients with PC has shown that they, in a considerable proportion have developed clinical signs and symptoms of OA3. Thus, it can be speculated that when PC develops into OA this degeneration will primarily affect the disc margins. The rapid dissemination also to the attachments will affect the physical properties of these looser connective tissues. This will weaken the disc attachments which in turn will allow for a displacement of the disc of sufficient magnitude to cause CL. This hypothesis for the development of CL in PC does not preclude other mechanisms in single cases. Thus, one should expect that any degenerative lesion affecting the margins of the disc may also cause CL even when there is no previous history of PC. Our results may also indicate that any treatment should intervene early in the disease process of CL to prevent the development of a degenerative process. Acknowledgements. We wish to acknowledge Dr. Ainars Leonchicks and the A. Kirchenstein Institute (ASLA Ltd.) for performing the RT-PCR analyses.
References 1. Axelsson S, Bjo¨rnsson S, Holmlund A, Kopp S, Hjerpe A. Metabolic turnover of sulphated glycosaminoglycans and proteoglycans in temporomandibular joint cartilages with experimentally induced osteoarthrosis. Acta Odontol Scand 1994: 52: 65–71. 2. Axelsson S, Holmlund A, Hjerpe . An experimental model of osteoarthrosis in the temporomandibular joint of the rabbit. Acta Odontol Scand 1992: 50: 273–280. 3. DeLeeuw R, Boering G, van der Kuul I, Stegenga B. Hard and soft tissue imaging of the temporomandibular joint 30 years after diagnosis of osteoarthritis and internal derangement. J Oral Maxillofac Surg 1996: 54: 1270–1280. 4. Dobra K, Anda¨ng M, Syrokou A, Karamanos NK, Hjerpe A. Differentiation of mesothelioma cells is influenced by the expression of proteoglycans. Exp Cell Res 2000: 258: 12–22. 5. Eriksson L, Westesson P-LL., Rohlin M. Temporomandibular joint sounds in patients with disc displacement. Int J Oral Maxillofac Surg 1985: 14: 428–436. 6. Gulyas M, Dobra K, Hjerpe A. Expression of genes coding for proteoglycans and Wilms’ tumour susceptibility gene 1 (WT1) by variously differentiated benign human mesothelial cells. Differentiation 1999: 65: 89–96. 7. Maroudas AI. Balance between swelling pressure and collagen tension in normal and degenerate cartilage. Nature 1976: 260: 808–809. 8. Paegle DI, Holmlund AS, Hjerpe A. Matrix glycosaminoglycans in the temporomandibular joint disc in patients with painful clicking and chronic closed lock. Int J Oral Maxillofac Surg 2003: 32: 397– 400. 9. Stegenga B, De Bont LGM, Boering G. Osteoarthrosis as the cause of craniomandibular pain and dysfunction: a unifying concept. J Oral Maxillofac Surg 1989: 47: 249–256. 10. Theocharis AD, Tsolakis I, Hjerpe A, Karamanos NK. Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V0. Atherosclerosis 2001: 154: 367–376. 11. Wei L, Svensson O, Hjerpe A. Proteoglycan turnover during development of spontaneous osteoarthrosis in the guinea pigs. Osteoarthritis Cartilage 1998: 6: 410–416. Address: Anders Holmlund Department of Laborative Medicine Division of Pathology Karolinska Institutet Huddinge University Hospital Huddinge Sweden Tel: +46 85 858 3953 Fax: +46 8 774 3875 E-mail: [email protected]